Skip to content
Login Register

#TALENs

6 public questions tagged with this topic.

Transgenic Plant -2

Genome editing method most precise at predetermined site is:

Precision of genome editing methods varies drastically correlating with targeting mechanism. Random mutagenesis approaches such as TILLING based on EMS alkylation of guanine causing G to A transitions genome-wide at ~1 per 150 kb requires screening massive populations via CEL1 nuclease assay and mapping; transposon mutagenesis with Ac/Ds or Mutator inserts at TA dinucleotides semi-randomly within genes, excision leaves footprint and instability; T-DNA insertion via Agrobacterium integrates preferentially into euchromatic gene rich regions at 5'-UTRs causing large deletions and chromosomal rear

Ref: Doudna Charpentier Science 2014 CRISPR precision; Voytas Plant Cell 2013 Editing comparison.

View Details
Transgenic Plant -2

Like ZFN, TALENs use which nuclease domain?

Like zinc finger nucleases, transcription activator-like effector nucleases utilize catalytic domain of FokI endonuclease for cleavage because DNA binding domain lacks nuclease activity. TALE repeats provide programmable anchoring but cannot cut phosphodiester backbone; fusion via flexible linker to C-terminal 196 aa FokI cleavage domain confers scission capability. FokI domain requires dimerization, so two TALEN monomers engineered to bind opposite strands in tail-to-tail orientation with spacer 12 to 21 bp allow FokI domains to associate forming active nuclease complex generating double-stra

Ref: Christian et al. Genetics 2010 186:757 TALEN FokI; Joung Sander Nat Rev Mol Cell Biol 2013 FokI.

View Details
Transgenic Plant -2

Most commonly used RVDs include NI, HD, NG and NN which recognize:

Four repeat variable diresidues commonly used in TALEN assembly define simple cipher: NI comprising asparagine isoleucine preferentially binds adenine forming hydrogen bond between Asn and N7, HD histidine aspartate specifies cytosine via Asp carboxyl hydrogen bond to cytosine exocyclic amine and salt bridge, NG asparagine glycine recognizes thymine via van der Waals contacts between glycine carbonyl and methyl group at 5 position, NN asparagine asparagine tolerates guanine and adenine due to bulky purine accommodation but later improved by NK asparagine lysine or NH asparagine histidine more

Ref: Cermak et al. Nucleic Acids Res 2011 39:e82 RVD NI HD NG NN; Bogdanove Voytas Science 2011.

View Details
Transgenic Plant -2

TALENs are derived from proteins of:

Transcription activator-like effector nucleases originate from plant pathogenic bacteria Xanthomonas genus comprising Xanthomonas oryzae pv. oryzae causing bacterial blight of rice and Xanthomonas citri causing citrus canker. Pathogen delivers effector proteins via type III secretion apparatus into plant cell cytoplasm mimicking eukaryotic transcription factors: N-terminal type III secretion signal, central repeat domain 12-30 tandem repeats of 33-35 amino acids differing at two positions conferring DNA specificity, C-terminal activation domain and nuclear localization signals. Once in nucleus

Ref: Boch Science 2009 326:1509 TALE Xanthomonas; Bogdanove Curr Opin Microbiol; Moscou Bogdanove 2009.

View Details
Gene Tar getting

Which feature is common between TALENs and ZFNs?

Zinc finger nucleases and transcription activator-like effector nucleases are chimeric nucleases sharing cleavage module. Both fuse sequence specific DNA binding domain to non-specific FokI endonuclease catalytic domain from Flavobacterium okeanokoites. FokI must dimerize to cut DNA, so two ZFN or TALEN monomers bind opposite strands with spacer. DNA binding domains differ: zinc fingers versus TALE repeats, but nuclease domain identical. CRISPR instead uses Cas9 nuclease. Conservation of FokI domain explains similar requirements for FokI dimerization and spacing rules in both platforms.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details
Gene Tar getting

Which genome editing platform uses protein-DNA interactions to determine specificity?

Genome editing specificity can derive from protein-DNA contacts versus RNA-DNA base pairing. CRISPR Cas9 relies on guide RNA pairing and PAM. RNA interference uses small RNA-mRNA complementarity. Zinc finger nucleases use engineered arrays of Cys2His2 zinc finger domains, each recognizing three nucleotides via alpha helix contacting major groove. Tandem fingers create specific protein-DNA interface fused to FokI nuclease. Thus ZFN specificity is entirely determined by protein side chain interactions with DNA bases, requiring modular assembly and selection to achieve desired target without RNA

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details